Factor VIII (antihemophilic factor) isolated from plasma or commercial concentrates consists of multiple polypeptides with M.sub.r approximately 80,000-210,000. Additionally, recombinant DNA clones have allowed the construction of plasmids which direct the expression of Factor VIII protein in transfected mammalian cells. Further, recombinant DNA clones have allowed the construction of plasmids which direct the expression of fusion products of Factor VIII protein in transfected mammalian cells. Factor VIII:C, as it relates to the present invention, refers to a Factor VIII protein that can function to correct the hereditary bleeding disorder termed hemophilia. Factor VIII can be from a plasma derived or genetically engineered source, or fusion products thereof. Plasma derived Factor VIII could be of human, porcine, or bovine origin and concentrates thereof. The term Factor VIII is not meant to be a limitation but refers to a functional protein for treating bleeding disorders.
A plurality of methods have been used for separating Factor VIII:C from plasma. Tuddenham et al, JOURNAL OF LABORATORY CLINICAL MEDICINE, Vol. 93, p. 40 (1979), reported a one step method for separating Factor VIII:C from most other plasma proteins using polyclonal antibodies to von Willebrand factor coupled to agarose beads. Austen, BRITISH JOURNAL OF HAEM., Vol. 43, p. 669 (1979), purified Factor VIII employing an aminohexyl-substituted agarose. Zimmerman et al, U.S. Pat. No. 4,361,509 and U.S. Pat. No. Re. 32,011, describes a method of separating the component molecules of Factor VIII/von Willebrand factor (vWf) complex using a two step procedure, (1) adsorbing a VIII:C/vWf complex onto particles bound to a monoclonal antibody specific to vWf, and (2) adsorbing the eluted VIII:C on an aminohexyl agarose column for concentration and further purification of Factor VIII:C. Purification of Factor VIII:C from genetically engineered DNA clones has been achieved by first adsorbing the Factor VIII:C onto DEAE-Sepharose, eluting Factr VIII:C and passing the material through a Factor VIII monoclonal antibody column, Gitschier et al, NATURE, Vol. 312, Nov. 22, 1984, pp. 330-337. Rotblat et al, Biochemistry, 1985, 24, 4294-4300 describe a method for purifying human Factor VIII:C from cryoprecipitate, using a polyelectrolyte procedure as a preliminary step before immunoaffinity chromatography with antibody to von Willebrand factor followed by adsorption to a monoclonal antibody specific to Factor VIII:C. To date, the most successful purifications of Factor VIII:C from plasma and recombinant DNA clones have been accomplished by using monoclonal antibodies specific to either Factor VIII:C or von Willebrand factor.
Although monoclonal antibodies have been successfully used to obtain a relatively pure Factor VIII:C preparation, monoclonal antibodies can be present in the final preparation because of leaching from the support matrix. This raises the possibility of antigenicity when the final preparation is introduced into animal systems, since murine monoclonal antibodies are not a normal metabolite and are known to be antigenic. A second disadvantage of the use of monoclonal antibodies is the requirement of cell culture facilities for producing monoclonal antibodies and the concomitant cost and handling/processing associated with monoclonal antibodies.
This invention avoids the problems associated with monoclonal antibodies yet still provides a highly purified preparation using at least one phospholipid coupled to a support structure with the predominant phospholipid being phosphatidylserine.
Binding of Factor VIII:C to phospholipid vesicles has been demonstrated using an IRMA assay, sucrose gradient ultracentrifugation and column chromatography. Lajmanovich et al, Biochimica et Biophsica Acta, 678 (1981), 132-136, investigated the interaction between purified human Factor VIII and phospholipid vesicles. An equimolor mixture of PS and PC were studied by sucrose gradient ultracentrifugation. Other experiments, not shown in the reference, referred to the use of PS or PC vesicles. The authors showed that in the presence of PS/PC or PS/PE vesicles Factor VIII activity was completely separated from vWf using sucrose-density-gradient ultracentrifugation, VIII:C appearing with phospholipids at the top and vWf migrating to the bottom.
Yoshioka et al, Brit. J. Haem., 1983, 55, 27-36, studied the interaction between Factor VIII clotting antigen (VIII:C Ag) and phospholipid (PL) using an IRMA approach. Treatment of Factor VIII concentrate with purified phospholipids showed greatest VIII:C Ag reduction with PS, less with PE and very little with PC.
Andersson and Brown demonstrated by sucrose gradient ultracentrifugation that Factor VIII/von Willebrand factor binds to phospholipid vesicles in solution (BIOCHEM. J. [1981] 200, 161-167). The phospholipid vesicles were a 50/50 mixture of phosphatidylserine and phosphatidylethonalamine (PE) or a 50/50 mixture of phosphatidylserine and phosphatidylcholine (PS). Andersson et al were also successful at coupling phospholipid vesicles containing phosphatidylserine and phosphatidylethanol amine to cyanogen bromide-activated agarose gels, (THROMBOSIS RES. 23:481-489, 1981). A 50/50 mixture of PS/PE was again used. Factor VIII/von Willebrand factor was shown to bind to the phospholipid vesicles in the absence of Ca.sup.++ but attempts to elute any Factor VIII with 1M NaCl were unsuccessful. Factor VIII was eluted with 1M KSCN resulting in fairly low recovery of Factor VIII activity due to the lability of Factor VIII in KSCN.
A primary object of this invention is to obtain a relatively pure Factor VIII:C preparation from plasma derived or genetically engineered sources and fusion products thereof without the problems associated with monoclonal antibody purification.
Another object of this invention is to provide a purification process that can be performed in one step to purify Factor VIII:C and fusion products thereof directly from tissue culture fluids.
Yet another object of this invention is to use a phospholipid column that is predominantly phosphatidylserine, in combination with monoclonal antibodies for further purification and isolation of Factor VIII:C.